Dry reagent analytical devices typically involve absorbent pads in which there are disposed reagent systems which react with analytes in fluid test systems applied to the device to provide a detectable response. Certain of these devices involve an enzymatic reaction with the analyte in the presence of a peroxidase and a hydroperoxide to cause a detectable color change in a redox dye. These systems are normally based on the use of filter paper as the absorbant pad. Other such devices operate on the basis of immunoreactivity of a labeled antibody located in the reagent device which specifically binds with analyte in the test sample to provide a detectable response in a specified region of the test device. Nitrocellulose is a preferred base material for this sort of device due to its flow through properties.
These dry reagent devices are inexpensive and convenient to use but suffer from certain limitations. For example in the analysis of serum for the mb isoform of creatine kinase (CKMB) there has been observed poor immunochemical separation, i.e. the ability of antibodies to capture the analyte of choice without non-specific binding. The slower the separation, the more time analytes have to be captured. With faster separation there is a limited time for the reaction to take place. Dry reagent tests for urine creatinine sometimes exhibit instability due to incompatible chemicals.
There are many examples of incompatible chemicals in dry reagent systems. For example, the base in white blood cell reagents causes premature hydrolysis of protease substrate, iron in occult blood reagents causes premature oxidation of redox dye indicators to their colored form which is also the result of the presence of iodate in glucose reagents. In the case of copper based tests for creatinine, the copper can oxidize redox indicators such as tetramethylbenzidine to their colored form in the absence of creatinine.
Tests for occult blood in urine can be skewed by the presence of ascorbate in the urine test sample which acts as a reducing agent causing false negative results and urine protein tests can be rendered inaccurate by the presence of buffers in the urine sample being tested. Dry assay devices for determining white blood cells in urine can be influenced by interference due to proteins in the urine sample and whole blood assays, such as blood glucose and blood CKMB, suffer from interference caused by red blood cells. The present invention provides a means for alleviating these problems by overcoating the dry reagent device with a permeable transparent membrane.
Previous methods for dealing with these problems have involved separating the reagents into discrete, stacked layers. There are, however, problems associated with the use of the discrete, stacked layer configuration. Thus, the top layer(s) must allow the test sample and analyte to pass to the lower layers while continuing to separate certain interfering chemicals and/or biochemicals. For example, metals such as copper or iron should be separated from redox indicators and bases from protease substrates. Oxidants such as iodate and reductants such as ascorbate need to be separated from redox indicators such as tetramethylbenzidine. In addition, the top layer must be translucent to permit reading of the signal produced in the bottom layer. Nylon mesh and glass fiber paper are among the materials used as semitransparent, discrete layers. The use of these materials requires formats to hold the layers together, which formats are typically difficult to manufacture, do not provide complete contact between the layers and may not effectively prevent migration of reagents between the layers.
There are various diffusible, adhesive compositions which can be used to secure two layers in integrated, multilayered reagent devices. In U.S. Pat. No. 5,110,550 there is described an absorbent covering layer which is non-transparent until liquid is transported from a color forming area. In U.S. Pat. No. 3,992,158 a transparent support at the bottom layer of a multiple stack of layers is described. The support layer is not permeable, so this type of device is read from the bottom. U.S. Pat. No. 4,587,099 describes the use of a transparent plastic netting which is fixed to a polystyrene strip handle which serves to hold the reagent layers together. The device is read from the top and the netting is permeable. However, the permeable layer is not attached to the underlying reagent layer.
U.S. Pat. Nos. 4,806,311 and 4,446,232 describe the advantages of the use of multilayered reagent devices for carrying out immunoassays and in U.S. Pat. No. 4,824,640 there is disclosed a transparent reagent layer for analytical reagents which consists of a water soluble or water swellable component and an essentially water insoluble film forming component.